FasL基因siRNA表达载体的构建及其在肺癌A549细胞中的表达

Construction of FasL siRNA expression vector and its expression in lung cancer cell line A549

背景与目的Fas/FasL是肿瘤坏死因子超家族成员,与肿瘤细胞的凋亡有关。激活的淋巴细胞由于FasL表达上调对于Fas/FasL凋亡途径非常敏感,从而可能被肿瘤细胞反杀伤攻击,使激活的淋巴细胞被清除掉。为研究FasL基因的功能及肺癌的基因治疗,构建了针对FasL基因的siRNA的表达载体,观察转染细胞A549后其对激活的T淋巴细胞的反杀伤作用。方法利用网站选择适当位点设计并合成针对FasL基因mRNA的寡核苷酸序列,插入质粒pGCsi-U6中形成重组载体pSi-FasL。重组载体经测序鉴定后转染肺癌细胞A549,Western blot检测转染前后FasL蛋白的表达情况。结果测序结果与所合成的寡核苷酸完全一致,证实成功构建了针对FasL基因的siRNA表达载体。Western blot检测显示转染肺癌细胞A549后有效抑制了FasL基因的表达。结论针对FasL基因的siRNA表达载体成功构建,并能有效的抑制肺癌细胞A549中FasL基因的表达。

Background and objective Fas/FasL is a member of Tumor Necrosis Factor （TNF） super family,and related to tumor cell apoptosis. It is hypothesis by forward study that activated lymphocytes is more sensitive with Fas/FasL due to up-regulation of FasL expression, so it can be inverse killedly and cleared by tumor cell. The aim of this investigate is to study the fuction of FasL gene and gene therapy of lung cancer by to down-regulationg the FasL gene expression with a siRNA expression plasmid in lung cancer cell A549 as well as its inverse killing effect between activated T lymphocytes and lung cancer cell A549. Methods Potential RNAi oligonucleotides of FasL was designed and synthesized according to appropriate web site. Then a FasL siRNA plasmid was constructed using a pGCsi-U6 vector.The plasmid was sequenced to confirm the inserted sequence. Western blot analysis was used to assess the levels of FasL proteins after the constructed plasmids have been transfected into A549 cells. Resuits It was confirmed by sequencing that the plasmid was constructed successfully. The result of Western blot clearly showed that FasL siRNA plasmid inhibited FasL expression in A549 cells. Conclusion The construct of FasL siRNA plasmid is successful. FasL protein expression of A549 cell is effectively inhibited by RNAi .